Liposomal-polyene preliposomal powder and method for its preparation

ABSTRACT

A method is disclosed for preparing a stable preliposomal powder which, when reconstituted with water or saline solution, forms a suspension of liposomes containing a polyene drug, such as nystatin. The method involves the steps of combining at least one phospholipid with a first organic solvent to form a first solution, adding a clarifying amount of water to the first solution, combining a polyene with a second organic solvent to form a second solution, combining the first and second solutions to produce a substantially clear combined solution, and then removing the organic solvents, leaving a powder.

BACKGROUND OF THE INVENTION

The present invention relates to a method of preparing a freeze-driedpowder, which can be used to quickly and conveniently generate aliposomal formulation of a polyene such as nystatin.

Nystatin is a tetraene-diene polyene antibiotic, first isolated fromStreptomyces noursei, which is used in the treatment of various fungalinfections. Unfortunately, nystatin, as well as other polyenes, is notgenerally useful for parenteral administration, because of its hightoxicity and insolubility in water. A formulation of a polyene, such asnystatin, which could be administered parenterally would substantiallyenhance the drug's therapeutic usefulness.

Some of the inventors of this patent previously discovered that theseproblems could be overcome by formulating nystatin in phospholipidvesicles, or liposomes. Such a liposomal formulation is considerablyless toxic to the animal to which it is administered, but is stilleffective against fungal infection, and therefore is suitable forsystemic use. U.S. Pat. No. 4,812,312 discloses that invention, and isincorporated here by reference.

One drawback to some liposomal drug formulations is theirless-than-desirable shelf life. Another drawback is the relativecomplexity of the process needed to prepare them. In view of thesedrawbacks, it would be highly desirable to produce a stable, dryformulation which could be rehydrated when needed for treatment of apatient. Lyophilized, or freeze-dried, powders are a possible answer tothis need. However, in order to be practical, a lyophilized powder mustnot only be stable and capable of reconstituting as liposomes, it mustcapable of being prepared by a process that is simple and inexpensiveenough so that it will be practical and cost-effective for commercialuse.

The present invention solves these and other problems found in the priorart.

SUMMARY OF THE INVENTION

The present invention generally concerns a method for producing a powdersuitable for the preparation of polyene-containing liposomes uponsuspension in an aqueous solution. In one aspect, the present inventionrelates to a method of preparing a liposomal-polyene preliposomalpowder, comprising the steps of combining at least one phospholipid witha first organic solvent to form a first solution; combining the firstsolution with a clarifying amount of water, forming a clarified firstsolution; combining polyene with a second organic solvent to form asecond solution; combining the clarified first solution and the secondsolution to produce a substantially clear combined solution; andremoving substantially all the solvent from the combined solution. In apreferred embodiment of this aspect of the present invention, a methodof preparing a liposomal-nystatin preliposomal powder comprises thesteps of combining dimyristoyl phosphatidyl choline and dimyristoylphosphatidyl glycerol with t-butyl alcohol to form a first solution;combining the first solution with a clarifying amount of water, forminga clarified first solution; combining nystatin with dimethyl sulfoxideto form a second solution; combining the clarified first solution andthe second solution to produce a substantially clear combined solution;and removing substantially all the t-butyl alcohol and dimethylsulfoxide from the combined solution.

In another aspect, the present invention relates to a method ofpreparing a parenterally-administrable polyene preparation, comprisingthe steps of combining at least one phospholipid with a first organicsolvent to form a first solution; combining the first solution with aclarifying amount of water, forming a clarified first solution;combining polyene with a second organic solvent to form a secondsolution; combining the clarified first solution and the second solutionto produce a substantially clear combined solution; removingsubstantially all the solvent from the combined solution, therebyforming a preliposomal powder; and adding a pharmaceutically acceptablesolvent to the preliposomal powder.

In another aspect, the present invention relates to a liposomal-polyenepreliposomal powder prepared by a method comprising the steps ofcombining at least one phospholipid with a first organic solvent to forma first solution; combining the first solution with a clarifying amountof water, forming a clarified first solution; combining polyene with asecond organic solvent to form a second solution; combining theclarified first solution and the second solution to produce asubstantially clear combined solution; and removing substantially allthe solvent from the combined solution, leaving a preliposomal powder. Aparticular embodiment of this aspect of the present invention is alyophilized nystatin powder, comprising nystatin, dimyristoylphosphatidyl choline, and dimyristoyl phosphatidyl glycerol, where theweight ratio of DMPC to DMPG is about 7:3, and where the powder is freeof halogenated solvents. It is believed that the relative proportions ofwater and the first organic solvent affect the nature andcharacteristics of the resulting powder, including the way in which thepowder behaves when hydrated. The polarity of the solution, with theproportion of water preferably being no greater than the proportion ofthe first organic solvent, is believed to affect the organization of thematerials in solution, and thus the nature of the powder that isultimately formed.

In another aspect, the present invention relates to a substantiallyclear, filterable polyene solution, comprising at least one polyene, atleast one phospholipid, at least one organic solvent, and a clarifyingamount of water.

The present invention, in its various aspects, provides surprisingadvantages over the prior art. For example, it has been found thatmerely combining phospholipids with an organic solvent such as t-butylalcohol produces a solution that is not clear, and therefore is notdesirable for use in producing a preliposomal powder. The presentinvention makes use of the surprising discovery that a clarifying amountof water can be added to the solution of polyene and organic solvent,yielding a clear solution which is suitable for use in subsequent stepsof the method. The clarity of solution permits substantially uniformcontact between the polyene and the phospholipids in subsequent stages.Further, the filterability of this solution permits contaminatingmicroorganisms to be removed readily prior to lyophilization. Thislatter point is particularly important with respect to polyenes such asnystatin, which would not tolerate autoclaving as an alternate means ofremoving microorganisms.

It is also surprising that the clarifying amount of water needed for usein the method of the present invention can range from about 10% of theamount of the first organic solvent (e.g., t-butyl alcohol) upward. Itis known that in other alcohol-lipid solutions, such large amounts ofwater cannot be used without causing precipitation. For example,precipitation will result in a solution of egg phosphatidyl choline inethanol when the amount of water added exceeds 30% of the volume ofalcohol.

It is also surprising that a second organic solvent, such as dimethylsulfoxide, can be added to the clear solution containing the clarifyingamount of water in conjunction with the addition of polyene withoutcausing either the lipids or the polyene to precipitate. Again, thesurprising clarity of the final solution renders the solution morereadily filterable, and thus makes the overall process more advantageousand economical. Without wishing to be bound by any particular theory toexplain these surprising results, it is believed that the first organicsolvent, such as t-butyl alcohol, forms a solvation complex with thephospholipids, a complex that arranges itself in a micellularconfiguration such that it remains clear even in a vast excess of wateror solvent. It is also believed that the polyene arranges itself in acomplex that may be micellular in nature, in the presence of thephospholipids.

The present invention facilitates the formulation and reconstitution ofliposomal-polyene from a degradation-resistant preliposomal powder. Thesimplicity of the present invention makes it suitable for large-scalemanufacturing. Further, it produces a stable powder which can be easilystored for at least one year. In addition, when reconstituted, theproduct of the present method forms multilamellar liposomes which have amean size that is suitable for administration to humans, for example inthe systemic administration of liposomal nystatin to treat a fungal orviral infection.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

A method in accordance with the present invention can include thefollowing steps. First, one or more phospholipids are combined with afirst organic solvent. The phospholipids which can be used are thosewhich are suitable for the preparation of liposomes, and are well-knownto those skilled in the art. Two specific examples that are particularlypreferred in the present invention are dimyristoyl phosphatidyl cholineand dimyristoyl phosphatidyl glycerol. The preferred weight ratio ofDMPC:DMPG is approximately 7:3. Suitable organic solvents for use a thefirst organic solvent include t-butyl alcohol. The ratio of phospholipidto first organic solvent is preferably between about 10 g: 160 cc andabout 10 g:640 cc, and is most preferably about 10 g: 320 cc. Thecombination of phospholipid and first organic solvent creates a firstsolution.

A clarifying amount of water is then added to the first solution,resulting in the clarification of that solution. A "clarifying amount ofwater" is used in this patent to mean an amount of water that will beeffective to clarify the already-existing solution of phospholipid andfirst organic solvent. This amount of water is preferably equal to atleast 10% by weight of the amount of first organic solvent in the firstsolution. The clarifying amount of water can range up to 100% by weightof the amount of first organic solvent, and can go even higher. In apreferred embodiment, the volume ratio of water to t-butyl alcohol isabout 1:4. In other words, in a preferred embodiment, the ratio ofphospholipid to t-butyl alcohol to water is 10 g: 320 cc: 80 cc.

The next step is to combine at least one polyene with a second organicsolvent to form a second solution. The ratio of polyene to secondorganic solvent preferably ranges from about 1 g: 5 cc to about 1 g: 25cc. Suitable polyenes include nystatin, amphotericin B, filipin,hamycin, and mepartricin. Nystatin is particularly preferred. Suitablesecond organic solvents include dimethyl sulfoxide (DMSO) and dimethylformamide. In one preferred embodiment of the present invention, theratio of nystatin to DMSO is approximately 1 g: 7 cc.

The first and second solutions can be filtered separately before theyare combined, for example through a 0.45 μM pore size filter cartridge.However, any contaminants which are present, such as microorganisms, canusually be removed in a single filtration step immediately before theorganic solvents are removed from the combined solution. If the amountof contaminants present in the combined solution causes problems withfiltration, then additional filtration steps can suitably be added.

The clarified first solution is then combined with the second solutionto produce a substantially clear combined solution. Preferably, theconcentration of nystatin in the combined solution is about 2.5-2.75mg/ml and the concentration of phospholipid is about 25-27.5 mg/ml. Theratio of solids to liquid in this solution is believed to be importantto the ready reconstitution of the preliposomal powder into liposomeswhen water is added. If the solids concentration is too high, theresultant dry product is denser than optimal and does not perform aswell as desired on reconstitution.

Next, substantially all of the organic solvents are removed from thecombined solution, for example by lyophilization, producing apreliposomal powder. The powder can be reconstituted into an aqueousformulation of liposomal polyene by adding a pharmaceutically acceptablesolvent, such as water or saline solution.

The following is a specific example of how the process of the presentinvention can be performed. The quantities given below are for the sakeof example, and could be scaled up proportionately.

Seven g of dimyristoyl phosphatidyl choline and 3 g of dimyristoylphosphatidyl glycerol (Lipoid KG, Ludwigshafen, Germany) are transferredto a first mixing vessel. To this vessel, 250 cc of t-butyl alcohol areadded, and the solution is mixed. This first solution is cloudy at thispoint. Then, 250 cc of water are added and mixed until dissolved,resulting in a clarified solution. Nystatin (1.1 g, American Cyanamid,Pearl River, N.Y.) is separately transferred to a second mixing vessel,and 7 cc of dimethyl sulfoxide is added. The theoretical amount ofnystatin needed for this particular preparation is 1 g (1:10 weightratio of nystatin: phospholipid), but it is preferred to add 110% of thetheoretically needed amount. This second solution is mixed untildissolved, and is mixed with the first solution to produce a combinedsolution, which is clear yellow.

The combined solution is analyzed for nystatin concentration by aspectrophotometric assay. If needed to correct the concentration, anappropriate volume of a diluent, such as water, is added.

Next, the combined solution is passed through a sterile, 0.22 μM poresize membrane, and 20 cc of the filtrate is transferred into a 100 ccvial. A lyophilization stopper is then loosely placed on the filledvial, and the vial is placed into a sterile lyophilization chamber,where the solvents are removed. While the vial is placed into thechamber, a thermocouple probe is inserted into the vial, in order to beable to monitor its temperature during the freeze-drying process. When aplurality of vials are placed in the chamber, thermocouples are placedso that a reasonable cross-section of the chamber is monitored (e.g.,top, middle, and bottom of chamber).

The circulating fluid in the freeze-drying chamber is adjusted to -45°C. The chamber is then evacuated to a vacuum of no more than 200microns. The circulating fluid temperature is then ramped to 10° C. overa minimum of about 24 hours at an approximate rate of 2.3° C./hour. Thechamber vacuum is adjusted to sweep slowly from 60 to 100 microns usingN to control the sweep rate. When the coldest thermocouple reaches -5°C., the circulating fluid is ramped to 28° C. over a minimum of fourhours at an approximate rate of 5.5° C./hour. When the coldestthermocouple reaches 28° C., the product is at terminal dryingtemperature, and is held there for about 12-30 hours. At the end of thistime, the chamber is adjusted to atmospheric pressure using N and NFpassed through a microbiologically retentive filter. Prior to unloadingthe freeze-drying chamber, the temperature of the circulating fluid isadjusted to 25° C. At the end of the lyophilization cycle, the vials arefilled with nitrogen gas and the stoppers are fully closed. Thelyophilized powder appears pale yellow, and contains less than 2%residual t-butyl alcohol and less than 1% residual dimethyl sulfoxide.

The formulation is reconstituted by adding about 50 cc of water to thepowder for every 1 g of polyene. It is preferred to heat the solutionabove 27° C., most preferably between about 30° and 45° C. for about15-60 minutes, to aid the hydration process. The powder initiallydisperses into clumps several tens of μ in diameter. When the solutionis warmed, the clumps hydrate and spontaneously form liposomes. Thetemperature at which this transition occurs may be due to the phasetransition temperature of the lipids, which for the above-describedmaterials is around 23° C.

After reconstitution, the mean particle size is about 2-3 μM, with notmore than 1% having a diameter over 8 μM. The incorporation efficiencyof drug in liposomes is greater than 90%, and may approach 100%.

The preceding description is intended to illustrate the presentinvention. It is not intended to be an exhaustive list of all possibleembodiments of the invention. Persons skilled in this field willrecognize that modifications could be made to the description givenabove that would remain within the scope of the invention.

We claim:
 1. A method of preparing a liposomalpolyene preliposomalpowder, comprising the steps of:a. combining at least one phospholipidwith t-butyl alcohol to form a first solution; b. combining the firstsolution with an amount of water effective to clarify the firstsolution, forming a clarified first solution; combining polyene with anorganic solvent selected from the group consisting of dimethyl sulfoxideand dimethyl formamide to form a second solution; d. combining theclarified first solution and the second solution to produce asubstantially clear combined solution; and e. removing substantially allthe solvent from the combined solution by lyophilization.
 2. The methodof claim 1, where the amount of water effective to clarify the firstsolution is equal to at least 10% by volume of the amount of t-butylalcohol.
 3. The method of claim 1, where the organic solvent of step (c)is dimethyl sulfoxide.
 4. The method of claim 1, where the phospholipidsare dimyristoyl phosphatidyl choline an dimyristoyl phosphatidylglycerol.
 5. The method of claim 4, wherein the ratio of dimyristoylphosphatidyl choline to dimyristoyl phosphatidyl glycerol is about 7:3by weight.
 6. The method of claim 1, where the ratio of phospholipid tot-butyl alcohol is between about 10 g: 160 cc and about 10 g: 640 cc. 7.The method of claim 1, where the ratio of polyene to the organic solventof step (c) is between about 1 g: 5 cc and about 1 g: 25 cc.
 8. A methodof preparing a liposomalnystatin preliposomal powder, comprising thesteps of:a. combining at least one phospholipid with t-butyl alcohol toform a first solution; b. combining the first solution with an amount ofwater effective to clarify the first solution, forming a clarified firstsolution; c. combining nystatin with an organic solvent selected fromthe group consisting of dimethyl sulfoxide and dimethyl formamide toform a second solution; d. combining the clarified first solution andthe second solution to produce a substantially clear combined solution;and e. removing substantially all the solvent from the combined solutionby lyophilization.
 9. The method of claim 8, where the amount of watereffective to clarify the first solution is equal to at least 10% byvolume of the amount of t-butyl alcohol.
 10. The method of claim 8,where the organic solvent of step (c) is dimethyl sulfoxide.
 11. Themethod of claim 8, where the phospholipids are dimyristoyl phosphatidylcholine and dimyristoyl phosphatidyl glycerol.
 12. The method of claim11, where the ratio of dimyristoyl phosphatidyl choline to dimyristoylphosphatidyl glycerol is about 7:3 by weight.
 13. The method of claim 8,where the ratio of phospholipid to t-butyl alcohol is between about 10g:160 cc and about 10 g: 640 cc.
 14. The method of claim 8, where theratio of polyene to the organic solvent of step (c) is between about 1g: 5 cc and about 1 g: 25 cc.
 15. The method of claim 8, where thevolume ratio of water to t-butyl alcohol is about 1:4.
 16. The method ofclaim 8, where the concentration of nystatin in the combined solution isabout 2.5 mg/ml.
 17. The method of claim 8, where the concentration ofphospholipid in the combined solution is about 25 mg/ml.
 18. A method ofpreparing a liposomal-nystatin preliposomal powder, comprising the stepsof:a. combining dimyristoyl phosphatidyl choline and dimyristoylphosphatidyl glycerol with t-butyl alcohol to form a first solution; b.combining the first solution with an amount of water effective toclarify the first solution, forming a clarified first solution; c.combining nystatin with dimethyl sulfoxide to form a second solution; d.combining the clarified first solution and the second solution toproduce a substantially clear combined solution; and e. removingsubstantially all the t-butyl alcohol and dimethyl sulfoxide from thecombined solution by lyophilization.
 19. The method of claim 18, wherethe amount of water effective to clarify the first solution is equal toat least 10% by volume of the amount of t-butyl alcohol.
 20. The methodof claim 18, where the ratio of nystatin to dimethyl sulfoxide isbetween about 1 g: 5 cc and about 1 g: 25 cc.
 21. The method of claim18, where the ratio of dimyristoyl phosphatidyl choline and dimyristoylphosphatidyl glycerol to t-butyl alcohol is between about 10 g: 160 ccand about 10 g: 640 cc.
 22. The method of claim 18, where the volumeratio of water to t-butyl alcohol is about 1:4.
 23. The method of claim18, where the concentration of nystatin in the combined solution isabout 2.5 mg/ml.
 24. The method of claim 18, where the concentration ofDMPC and DMPG in the combined solution is about 25 mg/ml.
 25. A methodof preparing a parenterally-administrable polyene preparation,comprising the steps of:a. combining at least one phospholipid witht-butyl alcohol to form a first solution; b. combining the firstsolution with an amount of water effective to clarify the firstsolution, forming a clarified first solution; c. combining polyene withan organic solvent selected from the group consisting of dimethylsulfoxide and dimethyl formamide to form a second solution; d. combiningthe clarified first solution and the second solution to produce asubstantially clear combined solution; e. removing substantially all thesolvent from the combined solution by lyophilization, thereby forming apreliposomal powder; and f. adding a pharmaceutically acceptable solventto the preliposomal powder.
 26. The method of claim 25, where the amountof water effective to clarify the first solution is equal to at least10% by volume of the amount of t-butyl alcohol.
 27. The method of claim25, where the organic solvent of step (c) is dimethyl sulfoxide, and thephospholipid comprises dimyristoyl phosphatidyl choline and dimyristoylphosphatidyl glycerol in a weight ratio of about 7:3.
 28. The method ofclaim 26, where the volume ratio of water to t-butyl alcohol is about1:4.
 29. The method of claim 25, where the concentration of polyene inthe combined solution is about 2.5 mg/ml.
 30. The method of claim 25,where the concentration of phospholipid in the combined solution isabout 25 mg/ml.
 31. A method of preparing a parenterally-administrablenystatin preparation, comprising the steps of:a. combining at least onephospholipid with t-butyl alcohol to form a first solution; b. combiningthe first solution with an amount of water effective to clarify thefirst solution, forming a clarified first solution; c. combiningnystatin with an organic solvent selected from the group consisting ofdimethyl sulfoxide and dimethyl formamide to form a second solution; d.combining the clarified first solution and the second solution toproduce a substantially clear combined solution; e. removingsubstantially all the solvent from the combined solution bylyophilization, thereby forming a preliposomal powder; and f. adding apharmaceutically acceptable solvent to the preliposomal powder.
 32. Themethod of claim 31, where the amount of water effective to clarify thefirst solution is equal to at least 10% by volume of the amount oft-butyl alcohol.
 33. The method of claim 31, where the organic solventof step (c) is dimethyl sulfoxide, and the phospholipid comprises DMPCand DMPG in a weight ratio of about 7:3.
 34. The method of claim 33,where the volume ratio of water to t-butyl alcohol is about 1:4.
 35. Themethod of claim 31, where the concentration of nystatin in the combinedsolution is about 2.5 mg/ml.
 36. The method of claim 31, where theconcentration of phospholipid in the combined solution is about 25mg/ml.
 37. A method of preparing a parenterally-administrable nystatinpreparation, comprising the steps of:a. combining dimyristoylphosphatidyl choline and dimyristoyl phosphatidyl glycerol with t-butylalcohol to form a first solution; b. combining the first solution withan amount of water effective to clarify the first solution, forming aclarified first solution; c. combining nystatin with dimethyl sulfoxideto form a second solution; d. combining the clarified first solution andthe second solution to produce a substantially clear combined solution;e. removing substantially all the t-butyl alcohol and dimethyl sulfoxideform the combined solution by lyophilization, thereby forming apreliposomal powder; and f. adding a pharmaceutically acceptable solventto the preliposomal powder.
 38. The method of claim 37, where the amountof water effective to clarify the first solution is equal to at least10% by volume of the amount of the t-butyl alcohol.
 39. The method ofclaim 37, where the volume ratio of water to t-butyl alcohol is about1:4.
 40. The method of claim 37, where the concentration of nystatin inthe combined solution is about 2.5 mg/ml.
 41. The method of claim 37,where the concentration of phospholipid in the combined solution isabout 25 mg/ml.
 42. A liposomal-polyene preliposomal powder prepared bya method comprising the steps of:a. combining at least one phospholipidwith t-butyl alcohol to form a first solution; b. combining the firstsolution with an amount of water effective to clarify the firstsolution, forming a clarified first solution; c. combining polyene withan organic solvent selected from the group consisting of dimethylsulfoxide and dimethyl formamide to form a second solution; d. combiningthe clarified first solution and the second solution to produce asubstantially clear combined solution; and e. removing substantially allthe solvent from the combined solution by lyophilization, leaving apreliposomal powder.
 43. The preliposomal powder of claim 42, where theamount effective to clarify the first solution is equal to at least 10%by volume of the amount of t-butyl alcohol.
 44. The preliposomal powderof claim 42, where the organic solvent of step (c) is dimethylsulfoxide.
 45. The preliposomal powder of claim 42, where thephospholipids are dimyristoyl phosphatidyl choline and dimyristoylphosphatidyl glycerol.
 46. The preliposomal powder of claim 45, wherethe ratio of dimyristoyl phosphatidyl choline to dimyristoylphosphatidyl glycerol is about 7:3 by weight.
 47. A liposomal-nystatinpreliposomal powder prepared by a method comprising the steps of:a.combining at least one phospholipid with t-butyl alcohol to form a firstsolution; b. combining the first solution with an amount of watereffective to clarify the first solution, forming a clarified firstsolution; c. combining nystatin with an organic solvent selected fromthe group consisting of dimethyl sulfoxide and dimethyl formamide toform a second solution; d. combining the clarified first solution andthe second solution to produce a substantially clear combined solution;and e. removing substantially all the solvent from the combined solutionby lyophilization.
 48. The preliposomal powder of claim 47, where theamount of water effective to clarify the first solution is equal to atleast 10% by volume of the amount of t-butyl alcohol.
 49. Thepreliposomal powder of claim 47, where the organic solvent of step (c)is dimethyl sulfoxide.
 50. The preliposomal powder of claim 47, wherethe phospholipids are dimyristoyl phosphatidyl choline and dimyristoylphosphatidyl glycerol.
 51. The preliposomal powder of claim 50, wherethe ratio of dimyristoyl phosphatidyl choline to dimyristoylphosphatidyl glycerol is about 7:3 by weight.
 52. The preliposomalpowder of claim 47, where the ratio of phospholipid to t-butyl alcoholis about 10 g: 320 cc.
 53. The preliposomal powder of claim 47, wherethe volume ratio of water to t-butyl alcohol is about 1:4.
 54. Thepreliposomal powder of claim 47, where the concentration of nystatin inthe combined solution is about 2.5 mg/ml.
 55. The preliposomal powder ofclaim 47, where the concentration of phospholipid in the combinedsolution is about 25 mg/ml.
 56. A liposomal-nystatin preliposomal powderprepared by a method comprising the steps of:a. combining dimyristoylphosphatidyl choline and dimyristoyl phosphatidyl glycerol with t-butylalcohol to form a first solution; b. combining the first solution withan amount of water effective to clarify the first solution, forming aclarified first solution; c. combining nystatin with dimethyl sulfoxideto form a second solution; d. combining the clarified first solution andthe second solution to produce a substantially clear combined solution;and e. removing substantially all the t-butyl alcohol and dimethylsulfoxide from the combined solution by lyophilization.
 57. Thepreliposomal powder of claim 56, where the amount of water effective toclarify the first solution is equal to at least 10% by volume of theamount of t-butyl alcohol.
 58. The preliposomal powder of claim 56,where the ratio of nystatin to dimethyl sulfoxide is about 1 g:7 cc. 59.The preliposomal powder of claim 56, where the concentration of nystatinin the combined solution is about 2.5 mg/ml, the concentration ofphospholipid in the combined solution is about 25 mg/ml, and the volumeratio of water to t-butyl alcohol is about 1:4.
 60. A substantiallyclear, filterable polyene solution, comprising at least one polyene, atleast one phospholipid, t-butyl alcohol, and an amount of watereffective to clarify the solution.
 61. A substantially clear, filterablenystatin solution, comprising nystatin, at least one phospholipid,t-butyl alcohol, and an amount of water effective to clarify thesolution.
 62. The solution of claim 61, where the solution furthercomprises dimethyl sulfoxide.
 63. The solution of claim 61, where theamount of water effective to clarify the solution is equal to at least10% by volume of the amount of t-butyl alcohol.
 64. The solution ofclaim 61, where the solution is substantially free of halogenatedsolvents.
 65. The solution of claim 61, where the phospholipids aredimyristoyl phosphatidyl choline and dimyristoyl phosphatidyl glycerolin a weight ratio of about 7:3.
 66. The solution of claim 61, where thevolume ratio of water to t-butyl alcohol is about 1:4.
 67. The solutionof claim 61, where the concentration of nystatin in the solution isabout 2.5 mg/ml.
 68. The solution of claim 61, where the concentrationof phospholipid in the solution is about 25 mg/ml.